The c-Jun amino-terminal kinases (JNKsJ/stress-activated protein kinases (SAPKs) play a crucial role in stress responses in mammalian cells. The mechanism underlying this pathway in the hematopoietic system is unclear, but it is a key in understanding the molecular basis of blood cell differentiation. We have cloned a novel protein kinase, termed hematopoietic progenitor kinase 1 (HPKI), that is expressed predominantly in hematopoietic cells, including early progenitor cells. HPK1 is related distantly to the p21Cdc42/Ra'~-activated kinase (PAK) and yeast STE20 implicated in the mitogen-activated protein kinase (MAPK) cascade. Expression of HPK1 activates JNK1 specifically, and it elevates strongly AP-l-mediated transcriptional activity in vivo. HPKI binds and phosphorylates MEKK1 directly, whereas JNK1 activation by HPK1 is inhibited by a dominant-negative MEKK1 or MKK4/SEK mutant. Interestingly, unlike PAK65, HPK1 does not contain the small GTPase Racl/Cdc42-binding domain and does not bind to either Racl or Cdc42, suggesting that HPK1 activation is Racl/Cdc42-independent. These results indicate that HPK1 is a novel functional activator of the JNK/SAPK signaling pathway.

The fibroblast growth factors (FGFs) play key roles in controlling tissue growth, morphogenesis, and repair in animals. We have cloned a novel member of the FGF family, designated FGF-18, that is expressed primarily in the lungs and kidneys and at lower levels in the heart, testes, spleen, skeletal muscle, and brain. Sequence comparison indicates that FGF-18 is highly conserved between humans and mice and is most homologous to FGF-8 among the FGF family members. FGF-18 has a typical signal sequence and was glycosylated and secreted when it was transfected into 293-EBNA cells. Recombinant murine FGF-18 protein (rMuFGF-18) stimulated proliferation in the fibroblast cell line NIH 3T3 in vitro in a heparan sulfate-dependent manner. To examine its biological activity in vivo, rMuFGF-18 was injected into normal mice and ectopically overexpressed in transgenic mice by using a liver-specific promoter. Injection of rMuFGF-18 induced proliferation in a wide variety of tissues, including tissues of both epithelial and mesenchymal origin. The two tissues which appeared to be the primary targets of FGF-18 were the liver and small intestine, both of which exhibited histologic evidence of proliferation and showed significant gains in organ weight following 7 (sometimes 3) days of FGF-18 treatment. Transgenic mice that overexpressed FGF-18 in the liver also exhibited an increase in liver weight and hepatocellular proliferation. These results suggest that FGF-18 is a pleiotropic growth factor that stimulates proliferation in a number of tissues, most notably the liver and small intestine.

The function of the tumor suppressor protein p53 is modulated by post-translational events, primarily by phosphorylation. p53 is phosphorylated at multiple sites by a variety of protein kinases depending on the cellular environment. It has been suggested that serine 34 of mouse p53 is specifically phosphorylated by a stress-activated protein kinase in response to ultraviolet radiation. Since serine 34 is a major site of phosphorylation of mouse p53 in vivo and its specific protein kinase is still not definitively identified yet, we have examined the c-Jun N-terminal kinase 1 (JNK1) activity on p53 by expressing JNK1 in 293T cells. We show here that activated JNK1 phosphorylates mouse p53 specifically at serine 34 in vitro, while a dominanant-negative JNK1 mutant does not phosphorylate p53. More importantly, JNK1 associates with p53 in vivo, with or without activation, confirming that JNK1 is indeed a p53 kinase. Interestingly, activated JNK2 and JNK3 also phosphorylate serine 34 of mouse p53. Furthermore, JNK2 and JNK3 also associate with p53 in vivo, indicating that not only JNK1, but also JNK2 and JNK3 are p53 N-terminal serine 34 kinases. Phosphorylation of p53 by JNKs may play an important role in nuclear signal transduction in response to environmental stress or tumorigenic agents.

The p38 mitogen-activated protein kinases (MAPK) play a crucial role in stress and inflammatory responses and are also involved in activation of the human immunodeficiency virus gene expression. We have isolated the murine cDNA clones encoding p38-␦ MAPK, and we have localized the p38-␦ gene to mouse chromosome 17A3-B and human chromosome 6p21.3. By using Northern and in situ hybridization, we have examined the expression of p38-␦ in the mouse adult tissues and embryos. p38-␦ was expressed primarily in the lung, testis, kidney, and gut epithelium in the adult tissues. Although p38-␦ was expressed predominantly in the developing gut and the septum transversum in the mouse embryo at 9.5 days, its expression began to be expanded to many specific tissues in the 12.5-day embryo. At 15.5 days, p38-␦ was expressed virtually in most developing epithelia in embryos, suggesting that p38-␦ is a developmentally regulated MAPK. Interestingly, p38-␦ and p38-␣ were similar serine/threonine kinases but differed in substrate specificity. Overall, p38-␦ resembles p38-␥, whereas p38-␤ resembles p38-␣. Moreover, p38-␦ is activated by environmental stress, extracellular stimulants, and MAPK kinase-3, -4, -6, and -7, suggesting that p38-␦ is a unique stress-responsive protein kinase.

Nuclear factor B (NF-B) and the Rel family of proteins are pleiotropic transcription factors that play central roles in the immune and inflammatory responses, as well as apoptosis. Here, we identified a serine/threonine protein phosphatase X (PPX; also called protein phosphatase 4 (PP4)) that specifically associated with c-Rel, NF-B p50, and RelA. The amino acid sequences of human and mouse PPX are 100% identical, and the PPX gene was mapped to human chromosome 16 p11.2. Overexpression of PPX, but not catalytically inactive PPX mutants, stimulated the DNA-binding activity of c-Rel and activated NF-B-mediated transcription. These results suggest that PPX is a novel activator of c-Rel/NF-B.The Rel/NF-B 1 family of transcription factors includes RelA (also called NF-B p65), RelB, c-Rel, NF-B1 p50 (also called NF-B p50), and NF-B2 p52 and is involved in immunological responses, cellular proliferation, and programmed cell death (1, 2). Rel/NF-B family members share the 300-amino acid Rel homology domain in their amino-terminal regions. They activate gene expression by binding to B sites via the DNAbinding domain located within this Rel homology region. Although the regulation of Rel/NF-B by its inhibitor IB has been extensively studied, control of the phosphorylation state of Rel/NF-B remains unclear. Previously, we showed that c-Rel and RelA are involved in CD28-mediated signal transduction (3, 4). We also showed that c-Rel is both constitutively phosphorylated in T cells and that it is also inducibly phosphorylated following T-cell receptor plus CD28 costimulation in T cells (3). c-Rel knockout mice exhibit profound defects in T-cell function, including lymphokine (interleukin 2, interleukin 3, and granulocyte macrophage-colony stimulating factor) secretion and the T-cell proliferative response to T-cell receptor plus CD28 costimulation (1). To further study the signaling pathway leading to c-Rel activation, we searched for c-Rel interacting kinases or phosphatases using the yeast two-hybrid system. Here we identify the serine/threonine protein phosphatase X (PPX; also called protein phosphatase 4 (PP4)) that specifically associated with c-Rel and activated NF-B-mediated transcription. MATERIALS AND METHODSYeast Two-hybrid Screening-Yeast single-copy plasmids pPC62 and pPC86 (6) were kindly provided by Dr. D. Nathans; the yeast-selectable marker genes LEU2 and TRP1 were swapped between these two plasmids, resulting in a GAL4 transcriptional activation domain vector containing the LEU2 selectable marker (designated pTA), and a GAL4 DNA-binding domain vector carrying the TRP1 selectable marker (designated pDB). DNA encoding amino acids 2 to 300 of c-Rel was cloned into the GAL4 DNA-binding domain vector pDB, designated pDB-c-Rel, and used as bait to screen a human B lymphocyte cDNA library (CLON-TECH). Positive yeast clones were selected by colony filter ␤-galactosidase assay as described (6), and yeast DNA was recovered from positive clones and transformed into Escherichia coli. Plasmids containing cDNA clones were ...